Vol. 28, No. 4, p. 458-471
December, 1964
Copyright ( 1964 American Society for Microbiology
Printed in U.S.A.
Children's Hospital of Philadelphia, Department of Pediatrics, School of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania
..................................................................................... 461
Development of Inclusion Body .461
Chromosomal Aberrations .461
Reaction of Cells in Tissue Culture .461
..................................................................................... 463
Serological Epidemiology .463
Strain Differences .463
Precipitins .463
............................................................................. 464
Reactivation ................................................................................ 465
Persistent Infection in Tissue Culture......................................................... 466
Interferon.................................................................................... 466
Unusual Manifestations of Primary Infection in Newborn and Older Infants .467
Newborn.................................................................................. 467
Older infants............................................................................... 467
Mechanisms Whereby the Virus Reaches the Nervous System .467
Antiviral Chemotherapy in Herpetic Infection .467
The use of the word Herpes, as applied to
certain manifestations of skin disease, has a
long tradition, starting with Hippocrates. The
many interpretations of its meaning through 25
centuries were discussed in a scholarly review by
Beswick (9), who pointed out that the first
author to give a clear account of the disease
Herpes Febrilis was Richard Morton in 1694, but
that it was not until the end of the 19th century
1 A contribution to the Symposium on "Current
Progress in Virus Diseases" presented as part of
the program for the Centennial of the Boston City
Hospital, 1 June 1964, with Maxwell Finland serving as Consultant Editor, and John H. Dingle and
Herbert R. Morgan as moderators.
that the modern concept finally was accepted.
After the nomenclature of the disease was established, the causative agent, when isolated in
1912, came to be known as the Virus of Herpes
Simplex. In 1953 the taxonomy of viruses became
a subject of concern, and the virus received the
designation "Herpesvirus hominis" (2), a name
which has not received general acceptance in the
literature. It seems probable that, with the continued discovery of new viruses from other species,
which have the characteristics of the herpesvirus
of man, and with further study of viral structure,
a more useful nomenclature will be devised. [See
Melnick et al. (44) and Wildy (84).]
The study of the virus has suffered from the
interference of two world wars. World War I
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INTRODUCTION................................................................................. 458
.................................................................................... 459
............................................................ 459
Composition of the Virus .459
Changes Induced by Infection .460
Induction of Enzymes .460
Adsorption ................................................................................. 460
Penetration................................................................................... 460
Multiplication .............................................................................. 460
Release of Virus .461
VDOL. 28, 1964
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started just at the time that Gruter had demon- (56, 8). Surrounding the core is the stable capsid,
strated that the virus could be isolated from the which has the characteristics of an icosahedron
dendritic ulcer of man, and that it would cause a with a 5:3:2 axial symmetry. This measures
similar lesion in the rabbit. Communication was 1,000 A in diameter and is composed of 162
virtually shut off until 1919, when L6wenstein capsomeres, which are elongated structures, 90
repeated Grilter's work and extended it to the to 100 A in diameter by 120 to 135 A in length.
isolation of the same virus from the herpes vesicle To fit the symmetrical arrangement, 150 of
of the skin. As a supposed cause of Von Economo's them are hexagonal and 12 are pentagonal in
encephalitis, epidemic for about 10 years after cross section. Many virions have envelopes, deWorld War I, the virus was studied extensively rived from host-cell membrane, which surround
during the 1920's but this interest died down in the capsid and bring the size of the particle to
the 1930's, when it became clear that Von 1,450 to 2,000 A in diameter. As seen under an
Economo's disease was not caused by this virus. electron microscope, virions vary in the comAt this time, the role of the virus as a disease pleteness of their structure; there are particles
agent came in question, because it was found to complete with envelopes ("enveloped particles")
cause its recognized clinical manifestation of and a central core containing nucleic acid ("full"
"fever blisters" only in those individuals who particles); particles with envelopes but an empty
had circulating antibodies, and therefore did not central core ("empty" particles); and particles
act like an infectious agent in the usual sense. without envelopes ("naked" particles), some
The problem was clarified in 1939 when it was with "full" and some with "empty" cores (80).
demonstrated that the virus caused stomatitis, Sections of infected cells, fixed in osmium and
as a primary infection, in those without anti- embedded in Aquon, show a dense eccentric oval
bodies, while the recurrent disease of "fever or rod-shaped body, 350 A in width, lying in the
blisters" occurred only in those with circulating center (core) of the virion. This is known as the
antibodies after recovery from the primary in- nucleoid and is deoxyribonuclease-sensitive (19).
fection. This important milestone in virology The host nature of the envelope was shown
failed to excite major interest because of the chemically by Epstein and Holt (20), who
more pressing health problems imposed by World demonstrated the presence of adenosine triWar II. These early studies were considered in phosphatase both in the envelope and in the
extenso by Van Rooyen and Rhodes (79). Since host-cell membrane, and immunologically by
the end of the war, interest in the virus has Watson and Wildy (81), who reported the
increased rapidly. The work during the earlier agglutination of enveloped particles by antihost
postwar years was reviewed by Stoker (72) and serum and of naked particles by antiviral serum
Weisse (82). This review will be directed chiefly and not vice versa.
to a description of the fundamental aspects of
research on the virus. The following subjects
will be considered: structure, biochemistry,
Composition of the Virus
mechanism of cell infection, cytology, serology,
latent infection, and recent observations of
Russell et al. (60), in a preliminary report,
clinical interest.
described the composition of the virus as 100
parts DNA, 25 parts carbohydrate, and 320 parts
phospholipid, to 1,000 parts protein. The DNA
The introduction of the technique of negative is double stranded and denser than that of the
staining with phosphotungstic acid for electron host cell, 1.727 g/ml as compared with 1.710 g/ml
microscopy (10) provided a new understanding (57). It has a sedimentation constant of 44S and
of viral structure. Wildy and Watson (85) a molecular weight of 68 X 106. The bases of the
studied the morphology of the virus by use of viral DNA are arranged in different proportions
this technique. The virus particle ("virion") con- as compared with those of the host DNA; the
sists of a roughly spherical central "core" guanine-cytosine to adenine-thymine ratio is 68%O
measuring 750 A in diameter, within which is in viral DNA and 42 % in host DNA. Each
contained the virus nucleic acid, now firmly viral particle is estimated to contain 1.2 X 10-1o
established as deoxyribonucleic acid (DNA) /Ag of DNA (58).
Induction of Enzymes
Dubbs and Kit (17) studied the thymidine
kinase system in infected mouse fibroblast cells.
This enzyme, important in the phosphorylation
of thymidine, was found to be absent in a mutant
cell line, LM(TK -). When this cell line was infected with herpesvirus, the synthesis of thymidine kinase was induced. However, two mutants
of the infecting strain of herpes were found which
lacked this kinase-inducing activity.
Infection of the host cell and multiplication
and release of newly formed virus from the host
cell can be discussed in sequential steps.
Both "enveloped" and "naked" particles can
be adsorbed to the host cells. The "enveloped"
particles appear to be adsorbed more readily
than the "naked" particles (29). Smith (67)
produced evidence that only the "enveloped"
particles actually multiply in the HEp-2 cells
he apparently used. However, Wildy and Watson
(85), using a baby hamster kidney cell line
(BHK 21), calculated that the "naked" particle
must also be infectious. These authors also discussed the paradoxical situation wherein the
antiviral serum, which neutralizes the infectious
"enveloped" particles, cannot agglutinate them.
Epstein et al. (21) described the events that
follow attachment of the virus particle to the
host cell. The host cell villi enfold the particle,
which is drawn into a pinocytotic vesicle. It then
travels in a series of vesicles towards the nucleus,
during which time the "enveloped" particle is
gradually stripped of its envelope by a proteolytic
cell enzyme. [The paradox of destruction by a
host-cell enzyme of the envelope derived from
the host may be explained by the work of Roane
and Roizman (53), who demonstrated that there
was a change in antigenic structure of the hostcell membrane during the course of infection.
The envelopes therefore do not completely present
the original host-cell structure.] In the paranuclear area, the stripped particle is released
from the vesicle by some mechanism not yet
clear but which seems to be specific, since gold
particles, which are ingested in a similar fashion
to the virus particle, are not so released. The next
step has not been demonstrated but must be the
release of viral DNA, which then enters the
nucleus where replication, of new virus takes
The development of new virus has been studied
in sections of infected tissue.7The earliest particles
appear as small, dense spherical bodies, 300 to
400 A in diameter, known as "primary bodies"
(45). These are scattered through the nucleoplasm
and sometimes are arranged in a crystalline
array. These bodies correspond in size to the
nucleoid (see Structure). In the nucleus, these
acquire a single membrane which is considered
to be the protein capsid.
The central portion of the nucleoplasm appears,
from electron micrographs, to be the site of
viral multiplication; this has been confirmed by
Munk and Sauer (46). These investigators demonstrated that thymidine labeled with tritium given
at the same time as the virus was concentrated
in the central portion of the nucleus, whereas, if
the labeled thymidine was given 18 hr prior to
infection, the label was concentrated in the
subsequently marginated nucleoplasm. In the
nucleus, the primary particles enlarge and acquire
a membrane and are next seen in smooth-walled
vesicles near the nucleus, possibly dilatations of
the Golgi apparatus (19). The mechanism of
release of particles from the nucleus has not yet
been clarified. In the cytoplasm, the particles
appear to achieve maturity. It has been estimated
that one infected cell produces 1,000 new viral
particles, of which 5 to 10% are infectious (58).
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Changes Induced by Infection
After infection of cells in suspension, under
one-step growth conditions, the following biochemical changes were observed (59). Within the
first 2 hr, specific viral complement-fixing (CF)
antigens appear and there is an increase in the
activities of two enzymes (DNA nucleotidyl
transferase and deoxyribonuclease I) associated
with DNA synthesis. DNA precursors reach a
critical level by the fourth hour after infection,
and by the fifth hour "heavy" viral DNA can be
detected. The formation of this continues until
the seventh hour and then levels off. Viral particles, seen shortly after the appearance of DNA,
increase in number following a similar curve.
Infectivity, however, does not become detectable
until the sixth hour, after which it increases
rapidly until the ninth hour and then levels off.
VOL. 28, 1964
Infection with herpesvirus leads to a number
of different changes in the infected cell.
virus, but probably can be accounted for by the
excess of DNA that is made in infected cells over
and above that required for the manufacture of
viral particles (48). The virus particles appear,
in the otherwise homogenous A body, as spherical
solid or hollow granules (A granules). These
granules make their first appearance at the time
the infectious virus is detected, and they increase
in number as the amount of infectious virus
increases. They are absent in the late stages of
infection. They contain, as does the A body,
DNA, RNA, and a nonhistone protein. About
18 hr after infection, the A bodies completely fill
the nucleus; at this time, some RNP is extruded
from the nucleus to form cytoplasmic RNP bodies
(C bodies) in the region of the Golgi apparatus.
It is uncertain what these represent and whether
virus may be extruded at the same time, by the
same mechanism.
Development of Inclusion Body
This has been the hallmark of herpes infection
since it was first described by Lipshtitz in 1921,
Chromosomal Aberrations
and has been studied by several authors. The
Hamper and Ellison (26) in 1963 reported an
most recent and detailed study was by Love and
Wildy (41). They used Toluidine Blue-molybdate increased incidence of chromosome breaks in the
stain on HeLa cells infected with a high-multi- MCH line of Chinese hamster cells after infection
plicity inoculum, and described the series of with herpesvirus. Stich et al. (71), using a
events that follow the entrance of the virus into virulent and an attenuated strain of herpesvirus
the cell. The first change, which occurs within 30 in male and female diploid cells of the Chinese
min after infection, is an enlargement of the hamster and human embryonic lung cells, renucleoli due to the increase of ribonucleoprotein ported that gaps and breaks in the chromosomes
(RNP) bodies (nucleolini). These are subse- were seen early in the infection, while uncoiling
quently extruded from the nucleoli into the and fragmentation of chromosomes occurred
nucleoplasm to form RNP bodies, called B later. The virulent strain led to damage of 90%
bodies to distinguish them from the A inclusions of the chromosomes at the peak, which was
which develop later. By 3 hr after infection. reached by the twenty-eighth hour after inRNP from the pars amorpha begins to diffuse fection. They emphasized that the damage was
into the rarified nucleoplasm that surrounds the not randomly distributed but that the structural
nucleoli where, almost immediately, within 30 abnormalities were concentrated in "region 3" of
min, the classical inclusion (A body) begins as the X chromosome and "region 7" of chromosome
small masses of deoxyribonucleoprotein which no. 1, whereas changes in the other chromosomes
subsequently fuse. As the inclusion grows, it were not greater than those in the controls.
displaces the nucleoli until they disintegrate or
Reaction of Cells in Tissue Culture
become diffusely dispersed. It can be assumed
that the invading viral DNA both serves as a
The different cytological pictures, rounding
template for new DNA synthesis and induces the and giant cell formation, presented by HeLa
formation of messenger ribonucleic acid (RNA) and rabbit kidney cells infected with the herpesin the nucleoli. This determines the specific viruses isolated from patients, were described
structure of the viral protein and the induced first when these cells were reported to be useful
enzymes required for its formation. The viral for isolation purposes (61, 69). The possibility
protein is apparently synthesized in the nucleus, that the cytological reaction might be due to
since the first membrane (capsid) that surrounds different viral strains present in a mixed poputhe "primary body" can be seen in the nucleo- lation was first suggested by Tokumaru (73),
plasm under an electron microscope. The large working with Herpesvirus suis (pseudorabies).
DNA-containing inclusion body is not all new Evidence is accumulating that there are two
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Release of Virus
Infected HeLa cells are not disrupted by the
virus, but particles leak out of the cell by evaginating the membrane surrounding the vesicle or
that of the cell itself. Thus, they acquire a triplelayered envelope which, under an electron microscope, appears identical to that of the host-cell
membrane (19). It seems possible that with other
cell lines, e.g., BHK 21, virus disrupts rather than
leaks out of some of the infected cells so that
numbers of "naked" particles are also freed into
the medium (85).
could be separated by finding that there was more
rapid neutralization with the homologous than
with the heterologous serum (54).
Kohlhage (38) studied in detail the two types
of GC response described above. He found that
the "rR" strain had the same density as the
MP strain, namely, 1.2614 g/ml, while the "fR"
strain was lighter, 1.2539 g/mi. He also found
that "rR" strain was eluted from an ECTEOLA
column at a lower salt molarity than was the
"fR" strain. Combining the observations of
Roizman and Roane and Kohlhage, it would
appear as if the cytological reactions were based
on changes in surface structure of the virus
strains. The denser strains are associated with
the P-type reaction, and the somewhat lighter
strains are associated with the usual GC reaction;
the lightest strains produce the diffuse lytic reaction. The difference in density has been
speculatively ascribed to the lipid content (54).
The mechanism of the large syncytial giant
cell formation was studied microcinematographically by Barski and Robineaux (6). The infected
cell attracts to itself neighboring uninfected
cells, their membranes adhere and fuse, and the
uninfected nuclei develop inclusion bodies and
collect in the center of the cytoplasmic syncytium. The formation of the small giant cell,
seen in the strains that do not form syncytia,
was ascribed by Love and Wildy (41) to a partial
reconstruction of the mitotic distortion caused by
herpesvirus infection. They also suggested that
failure to incorporate a damaged chromosome
could result in nuclear budding or "amitotic"
division. Amitotic division was advanced as an
explanation by previous workers on the basis of
histological observations (24, 33).
The formation of the syncytial giant cell,
characteristically associated with the development of intranuclear virus, must depend on a
change in the surface membrane of the infected
cell so that it may adhere and fuse with its
neighbors. This change does not seem to be
governed by the multiplication of the virus in
the nucleus but by a separate mechanism,
since Nii and Kamahora (50) reported the
formation of giant cells without the development of intranuclear inclusions in mouse fibroblasts (L cells). The change followed massive
inoculation of the cells with a GC strain virus
inactivated by ultraviolet light, but did not
follow an inoculation of a nonsyncytial forming
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common forms of cell reaction, and a less common,
intermediate form, each of which is induced by a
separate strain of H. hominis. The cytological
pictures, first described by Gray et al. (24), have
been repeatedly observed with minor modifications (28, 49, 63). The cytological effects are as
follows. The cells become rounded and pile up as
loosely attached cells on the surface of the monolayer (P strain of Gray et al., mP strain of Hoggan
and Roizman); syncytial giant cells are produced
as the result of an infected cell's attracting and
fusing with surrounding uninfected cells; two
forms occur, a diffuse area of lysis in which
hundreds of nuclei are collected about the middle
of the area [fR strain of Kohlhage and Siegert
(39)], or large distinct giant cells, each containing
dozens of nuclei, attached to one another by
thin protoplasmic bridges (GC strain of Gray
et al., MP strain of Hoggan and Roizman, rR
strain of Kohlhage and Siegert); and simple
rounding of cells without piling up [NP strain of
Gray et al., I (intermediate) strain of Schneweis].
Small giant cells, with fewer than ten nuclei, are
occasionally seen accompanying both P (mP) and
NP (I) strains. The type of host cell infected by
these virus strains modifies somewhat the actual
cytological changes observed. Nevertheless, the
cytological change induced appears to be genetically controlled in that the strain of virus
breeds true in the same cell line for many generations. However, passage through heterologous
hosts or conditions of storage leads to a change
in the cytopathic effect, favoring the appearance
of the GC form.
When the characteristics of the GC and P
strains were measured by other common parameters, they could not be distinguished from
one another; their rates of adsorption and of
growth in rabbit kidney cells were the same, the
pock sizes on the chorioallantoic membrane
(CAM) were similar, as was the virulence in
mice and rabbits, and, by standard tests, there
was complete cross-neutralization between them
(65). However, Roizman and Roane demonstrated slight differences between their MP and
mP strains in HEp-2 cells. The mP strain is
slightly denser than the MP strain, 1.271 to
1.260 g/ml (55); is eluted from a brushite column
by 0.55 M sodium phosphate as compared with
0.60 M for MP strain; and, when the kinetics of
neutralization with specific mP and MP antisera
were measured, a mixture of the two strains
V OL. 28, 1964
virus similarly treated. They suggested that the
GC strain must contain something which affects
the cell membrane and which can act when the
cells are coated with inactive virus. Further
evidence of a specific mechanism for the syncytial
formation in HeLa cells was given by Munk and
Sauer (46), who found that mitomycin C prevented the development of intranuclear virus but
not the development of syncytial giant cells.
Serological Epidemiology
The serological reactivity of H. hominis has
been the object of study for many years in relation to diagnosis and epidemiological survey.
One of the largest surveys, a study of 352 sera
taken from subjects of all ages, was reported by
Yoshino et al. (89) in 1962. These authors confirmed the trends, noted in previous studies, that
babies less than 4 months old had higher antibody
levels than children above that age, and that
between the ages of 5 and 20 years the frequency
of sera which contained antibody increased.
During this period titers varied extensively,
whereas in patients over 20 years of age the
antibody titers tended to be high or absent, the
"all or none" phenomenon of the early workers.
Although in general there was a close parallelism
between neutralizing and CF antibodies, some
adult sera had high titers of neutralizing antibody
without any detectable CF antibody. On the
other hand, some sera of the young children contained no neutralizing antibodies but did contain
CF antibodies to the viral (V), but not the
soluble (S), antigen, which were heat-labile (60 C
for 5 to 30 min). Such heat-labile antibodies were
not found among older children or adults. On the
basis of these findings, the authors suggested
that the earliest infections by the herpesvirus
lead to the production of abundant CF antibodies,
most of which are comparatively heat-labile. It
is only after repeated exposures, as noted by
Buddingh et al. (12), that a persistent infection
develops which results in the usual finding in
adults in whom the CF and neutralizing antibodies quantitatively parallel each other. In the
absence of a persistent infection, the antibodies
drop or disappear between reinfections. Because
the CF antibodies tend to drop sooner than the
neutralizing antibodies, some adults could have
a positive neutralizing and a negative CF anti-
body titer, depending on the time the blood
happened to be drawn. Yoshino and Taniguchi
(88) demonstrated that rabbits produce neutralizing antibodies as early as the third day after
immunization or infection, which differ from those
produced later by their requirement of at least
5 hemolytic units of complement for their detection. Although the later antibodies could
neutralize the virus in the absence of complement, the addition of complement enhanced the
speed of neutralization. This seemed to hold
true also for human sera, in which the addition
of complement enhanced the neutralizing power
by about twofold. It is worth noting that the
techniques used by Yoshino and his co-workers
for these studies have many advantages in
economy and speed of handling. The use of the
1-day-old egg enables the presence or absence of
neutralizing antibodies to be detected in 5 days,
because in the presence of infectious virus the
blastoderm ceases to grow (87). The titration of
the neutralizing activity of each serum can be
performed on a chick embryo monolayer growing
in a single petri dish (77).
Strain Differences
With the use of the standard type of neutralization and CF techniques, it was questionable
whether there were serologically distinguishable
strain differences between viral isolates. The
early opinion was that the herpesvirus was
antigenically homogeneous but, gradually, differences in neutralization and complement fixation
titers began to be reported (30). The adaptation
of the technique of neutralization kinetics (43) to
a study of herpesvirus by Schneweis (64) in 1962
and Ashe and Scherp (3) in 1963 has provided a
clear picture of antigenic differences. Schneweis
was able to group his 30 strains into two antigenic
types, and Ashe and Scherp grouped their 15
strains into four antigenic types and a heterogenous group.
A further approach to this problem may be
the application of the gel diffusion technique for
the detection of precipitins. The production of
precipitins by H. hominis was reported by
Tokumaru and Scott (75). An antigen derived
from the sonic treatment of rabbit kidney cell
tissue culture, infected with a recently isolated
strain of herpesvirus, was used to challenge 222
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random human sera; precipitin bands, from one
to five in number, were detected in 70 sera.
These were from patients shown to be convalescent from infection with herpesvirus by standard serological methods. In a selected group of
paired sera, precipitin bands were found to develop in some, but not all, convalescent sera
velopment of a tuberculin-like skin test in recovered patients who were inoculated intradermally with heat-killed virus grown in embryonated hens' eggs. This has been confirmed by
many workers with antigens from various sources.
In 1953 Brown (11) reported a similar observation in guinea pigs. Further analysis of this
phenomenon was reported by Tokumaru (74) in
1963. In addition to confirming Brown's work,
he was able to differentiate by diethylaminoethyl
(DEAE)-Sephadex column chromatography three
fractions which varied in sensitizing potential.
(Fig. 2).
One fraction, eluted in 0.11 M NaCl, had a high
sensitizing ability but a low CF activity; another,
Hypersensitivity to herpesvirus has been recog- eluted in 0.27 M NaCl, had a high CF activity
nized since Nagler (47) reported in 1944 the de- and high sensitizing ability; the viral antigen,
(Fig. 1). Preliminary analysis of the antigen by
density-gradient centrifugation in cesium chloride
revealed a distinct viral band and at least four
distinguishable bands in the soluble fraction
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FIG. 1. Precipitin bands appearing in the convalescent serum collected after a primary herpetic infection.
The wells were numbered clockwise, with the topmost well as number 1 and the center well as number 7. First
pair: 1 = acute, 2 = convalescent serum. Second pair: S = acute, 4 = convalescent serum. Third pair:
5 = acute, 6 = convalescent serum; 7 = antigen well. Note: precipitin bands appeared only in the convalescent serum of the second patient.
VOL. 28, 1964
eluted in 0.35 M NaCi, had little sensitizing
ability but a high CF activity.
The fact of latent infection by H. hominis is
well known, but the mechanism is still far from
clear. The absence of a suitable laboratory model
has hindered the acquisition of knowledge in
this area, and only a few attempts have been
made to study the problem.
operated upon for the relief of the pain of trigeminal neuralgia. Within a few days after
section of a branch of the fifth nerve, an extensive
eruption of herpetic vesicles frequently appears,
which covers the area of the freshly denervated
skin. Such eruptions only follow immediately
after nerve section since, after a second operation,
the vesicles appear in the freshly denervated
area but spare the anesthetic area of skin that
had followed the previous section. It is a matter
I 32
- 1.280
I - 15
FIG. 2. Precipitin bands in agar diffusion plate. Diagram of precipitin bands after isodensity-gradien
centrifugation of Herpesvirus hominis antigens.
of speculation as to whether the virus lies occult
The existence of a latent infection of the brain in the nerve ganglion or in the skin. Although no
in rabbits has been recognized since 1938 (52). virus has been found in the ganglia, on the few
Rare reports have suggested that reactivation of occasions that they have been tested, this clearly
such a latent encephalitis can be produced in does not mean that it may not be there in an
rabbits by anaphylactic shock (23) or by intra- occult form. However, it is difficult to conceive
muscular injections of adrenalin (62). In relation how the virus would get to the denervated area
to the latter, it was speculated that a change in of skin to produce the virus-containing vesicles.
blood supply, producing tissue anoxia, disturbed It seems more probable that the virus lies occult
the balanced host-parasite relationship. Reacti- in the skin and is reactivated as a result of the
vation of herpetic keratoconjunctivitis in rabbits changes in tissue metabolism caused by the
has been reported in 7 of 19 attempts after the denervation (18). The common recurrent lesion
induction of Arthus phenomenon in the infected is triggered by some disturbance of tissue blood
healed eye (1). Reactivation, as a secondary supply, such as fever, ultraviolet light irradiation,
effect of blood supply on local tissue metabolism, or emotion. Following the viremia of the primary
is also suggested by the studies of patients infection, the widely distributed virus becomes
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Persistent Infection in Tissue Culture
How much the observations on persistent infection can be applied to man is uncertain, but
there are suggestive analogies to support the
speculations in the above section. The disappearance of the infectious virus from the
patient roughly coincides with the development
of circulating antibodies. The virus either is
destroyed or becomes occult. In tissue culture,
the standard method of producing a persistent
infection is by the addition of antibody to the
medium (83). The effect of change in tissue
metabolism on the establishment and reactivation
of a latent infection was presented by Pelmont
and Morgan (51). These authors showed that,
when HeLa cells were placed in a nutritionally
deficient medium, infection with herpesvirus did
not result in the expected cytopathic effect, nor
could virus be isolated by passage to fresh cells.
However, if the missing nutrients were restored
to the infected culture, after several passages in
the deficient medium, the virus became detectable
and caused the characteristic cytopathic effect.
Further evidence along this line could be inferred
from the experience of Coleman and Jawetz (16),
who found that a modified latent infection could
be produced in tissue-culture cells derived from
adenocarcinoma of the lung (Maben cells) with
one strain (Z strain) of herpesvirus at 31 C. At
this temperature, the culture could be kept
growing for 9 months, although virus could consistently be isolated on subculture. However,
raising the temperature of incubation to 34 C
led to rapid destruction of the culture.
Another mechanism contributing to the development of resistance of cells to destruction by
herpesvirus is the production of interferon. In
1961 Barski and Cornefest (4) reported that, when
a low cancer line of mouse cells (N2) was infected
with polyoma virus, a latent infection was
regularly produced and that these cells resisted
further infection both by polyoma and herpesviruses. This they attributed to the production of
a virus-inhibiting substance similar to interferon.
Glasgow and Habel (22) described a continuous
line of mouse embryo cells, persistently infected
with polyoma virus (carrier culture 23-P), which
partially resisted challenge by herpesvirus. After
infection with herpesvirus at low multiplicity, an
incomplete cytopathic effect resulted and cells
grew out, leading to a double carrier culture that
elaborated both polyoma and herpesvirus and
was resistant to reinfection by either virus. The
equilibrium between the growth of the viruses
and the resistance was unstable so that, when
the culture was "cured" of its polyoma infection,
it was immediately destroyed by the herpesvirus.
It was suggested that polyoma and herpesviruses
were weak producers of endogenous interferons
so that resistance was only achieved by the
additive effect of both viruses. A herpesvirus
carrier culture could be produced in polyoma-free
susceptible cells by adding sufficient exogenous
interferon. The production of interferon by the
herpesvirus in vivo was demonstrated by inoculating adult guinea pigs with herpesvirus intraperitoneally. These were completely resistant to
superinfection by polyoma virus inoculated by
the same route within 24 hr and partially, but
decreasingly, resistant up to 7 days (5). No
therapeutic effect of interferon has been observed
on the course of herpetic keratoconjunctivitis in
man (15). However, in rabbits, if ultravioletirradiated influenza (Lee) virus was applied to
one eye within 24 hr after infecting both corneas
with herpesvirus, and reapplied four times daily
for 4 days, the lesion on the treated eye was
much less marked than in the untreated eye,
presumably as the result of the interferon produced by the cells infected with the attenuated
influenza virus. No benefit was derived from
application of the Lee virus after an ulcer had
developed (76).
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occult in the presence of circulating antibodies.
The site of reactivation, resultant on local disturbances of tissue metabolism, may be a matter
of chance. The frequency of lesions on the face,
as opposed to elsewhere on the body, may be
accounted for by the greater exposure of the face
to exogenous changes involving blood supply to
the skin. Special circumstances may determine
the site of recurrence, e.g., the truck driver with
a recurrent lesion on the back of his thigh, or
the postoperative neurosurgical patient with an
airway, from whose tracheal secretions reactivated virus may be the source of herpetic whitlow
on the fingers of the attendant susceptible nurses
VOL. 28, 1%.4
Mechanisms Whereby the Virus Reaches the
Nervous System
Herpetic encephalitis characteristically occurs
in one of two forms. In one, the brain seems to be
affected as part of a generalized spread of infection, and the virus can be isolated from other
organs as well as the brain. In the other, the
brain is specifically infected, the other organs
being free from the virus. A possible explanation
of these, different findings may be derived from
the recent studies of Johnson (32). He reinvestigated the mechanism of infection of the nervous
system for which no entirely acceptable explanation had heretofore been advanced. By the
technique of fluorescent-antibody -staining, he
showed that virus inoculated extraneurally in
suckling mice was concentrated about the small
cerebral vessels, indicating that it reached the
brain via the blood stream. After intranasal
inoculation in the same host, virus could reach
the brain by several routes: by direct invasion of
the subarachnoid space with dispersion of the
virus in the cerebrospinal fluid, the same course
as taken after intracerebral inoculation; by invasion of the Schwann cells of the olfactory and
trigeminal nerves and centripetal growth from
cell to cell; by the blood stream. The virus
clearly does not grow along the nerve axons, as
has been the most popular but unproven theory
in the past. It would seem a reasonable speculation that the isolated central nervous system
infection could be via direct invasion, leading to
the meningeal form of infection, or by the blood
stream, leading to widespread infection of the
brain parenchyma, or by both.
Antiviral Chemotherapy in Herpetic Infection
The search for antiviral drugs continues in
many fields. In 1962 Kaufman (34) reported that
5-iodo-2'-deoxyuridine (IDU) could cure experimental herpetic keratoconjunctivitis in rabbits,
and could be successfully used in the treatment
of herpetic ocular infections of patients (37).
These reports led to widespread, uncontrolled
use of the drug, and to the suggestion that a
cure rate of 80 to 90% could be expected in the
patient with an acute ulcer, but that patients
with pathological changes in the deep structures
of the eye usually did not respond. Controlled
studies in this disease have been complicated by
the difficulties encountered in making an accurate
diagnosis and the serious course of the disease
in some patients, which may limit experimental
observation. Two such studies (31, 42) reported
no advantage, and one (14) reported a statistically
significant advantage from IDU treatment. The
author of the last paper used the drug in both
acute and chronic herpetic infection, noting improvement only in the patients with acute infections but none among an equally large series
of patients with chronic herpetic changes in the
eye. The most recent and accurately controlled
study was that of Hart et al. (27), who treated
19 patients with IDU and 13 with neomycin
drops, every hour during the day and every 2 hr
at night for 7 days. By using a table of random
numbers to assign the cases to the IDU or the
neomycin series, providing rigid criteria for ad-
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Unusual Manifestations of Primary Infection in
-Newborn and Older Infants
Primary herpetic infection in the newborn is
characteristically a serious disease, almost always
leading to wide dissemination and death; a
similar infection in older infants does not usually
lead to clinical evidence of systemic infection.
Contrasts to this usual clinical picture have been
reported recently.
Newborn. Wilson and Martini (86) described a
newborn infant with a proven primary herpetic
infection whose lesions appeared to be confined
to the skin and mucous membranes of the nose
and who recovered. The herpesvirus was cultured
from the nasopharynx. Scott and Tokumaru
observed a similar mild primary infection in an
infant born of a mother who was diagnosed
clinically as having vulval herpes shortly after
delivery of the infant. The infant ran a low-grade
fever between the 5th and 14th days, and developed three or four blisters on the right shoulder
and about the umbilicus on the 8th day after
birth. A virus of low virulence to rabbit kidney
cell tissue culture was isolated from the vesicle
on the shoulder on the 12th day. The infant
made a complete -recovery. Both mother and
infant showed, a significant rise in antibodies,
indicating a primary infection in both.
Older infants. Becker et al. (7) from South
Africa reported a high fatality rate from primary
herpetic infection in infants who were suffering
from typical Kwashiorkowr in their second year
of life.
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mitting the patients to the study, limiting the "ragged, naked and structurally imperfect." The
period of study therapy to 7 days, and having process of viral assembly was discussed by Smith
only three clinical ophthalmologists caring for (66), who has shown the presence of naked strings
the patients in one hospital, they were able to of DNA early in development of the virus.
avoid some of the difficulties recognized in the These appear to be folded and encased in the
other studies. Under these circumstances, there protein capsid, either by an orderly sequential
was a healing response of the ulcer in 74% of assembly of capsomere subunits or simultaneIDU-treated eyes and 15% of the neomycin- ously. The latter possibility was suggested by
treated eyes. Failures in treatment have been the ragged protein coats that are seen on the
ascribed to the presence of chronic damage, but earliest recognizable viral particles, which are not
also may be due to the emergence of resistant infectious, as compared with the physically
mutants. Such mutants have been reported as perfect capsids which can be seen when infectious
occurring in human infection (40). At least one of particles are found late in the cycle. The action of
these has been reported to be susceptible to cytosine arabinoside does not appear to be on the
another antiviral agent, cytosine arabinoside incorporation of cytosine in the DNA molecule
(35), which can produce healing of herpetic but on a later step in the formation of DNA,
keratoconjunctivitis in rabbits even when ap- possibly at the stage of polymerization.
This review has arbitrarily selected certain
plied after ulceration has begun (78). Steroids
with IDU have been reported to be beneficial aspects of research on herpesvirus for emphasis.
(36). The clinical effect of IDU therapy is probably The frequent publications concerning this virus
not due to failure of virus multiplication, since, in suggest that knowledge in this area is increasing
treated rabbits, despite absence of ulcer, virus rapidly, from which an advance in the general
can be recovered although in a reduced titer (30a). understanding of virus infection can be anticiThere do not appear to have been any controlled pated.
studies of the use of IDU in herpetic infections of
the skin, although it has been reported that
herpetic vesicles heal more promptly with IDU
(13, 25).
KILBOURNE. 1961. Induced reactivation of
The action of this drug is to inhibit viral reproherpes simplex virus in healed rabbit corneal
duction in tissue culture, but the exact mechanism
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self-inhibition effect in infected cultures.
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VOL. 28, 1964
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